Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-01-06
2001-08-28
Campbell, Eggerton A. (Department: 1656)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S091200
Reexamination Certificate
active
06280945
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a method of solid phase hybridization in which streptavidin or derivatives thereof are non-covalently coated on the solid phase. Streptavidin and its derivatives prove stable under high temperature, high stringency hybridization conditions.
BACKGROUND OF THE INVENTION
Nucleic acid based diagnostics is enjoying particularly rapid growth at a time which has seen the overall demand for diagnostic assays continue to rise. Undoubtedly, this growth has been hastened by the increased pressure on medical professionals to contain costs. In response, the medical establishment is placing a growing emphasis on early intervention and prevention. Accordingly, assays which alert clinicians to a disease susceptibility have engendered strong interest. Nucleic acid based diagnostic assays promise to play a leading role in the expanding market for diagnostic assays.
Perhaps the single most critical step in nucleic acid based diagnostic assays is the selective hybridization of an oligonucleotide to its complementary target. A particularly favored means of accomplishing this has been through the use of solid phase supports. Solid phase DNA probe hybridization typically involves the direct or indirect covalent attachment of a synthetic “capture” oligonucleotide to a solid phase. The capture oligonucleotide is brought in contact with a sample comprising DNA or RNA under stringent hybridization conditions to promote duplex formation between the capture oligonucleotide and a complementary nucleic acid in the sample. Covalent attachment procedures require multiple chemical steps (which effect yield) and special handling equipment for the solid phase. Thus, it is costly and troublesome to change the particular oligonucleotide attached to the solid support. Previously, the severity of these problems was partially overshadowed by the relatively high cost and labor-intensive assay formats then in use. Today, however, the high capacity, high throughput, automated assay systems demanded by clinical laboratories has drawn increasing attention to the issues of manufacturing cost and complexity.
Application WO 90/10717 describe an attempted solution to the problem. In that publication, a nucleic acid tail of known sequence is attached to a capture oligonucleotide and the complement to the tail portion is covalently attached to the solid phase. Each new capture oligonucleotide shares the same tail such that the same solid phase can be used for all products. However, this approach requires that the tail oligo-complement hybrid be stable under the conditions of the sample hybridization. Since the sample hybridization conditions may vary with each new sample target, this approach requires a long region of complementarity to accommodate the high melting temperatures of possible target-oligonucleotide hybrids. Unfortunately, these long oligonucleotides have an increased risk of binding to non-target nucleic acids. Moreover, the secondary structural characteristics of long single strand nucleic acids can hinder access to the target.
Holodniy et al. (
Biotechniques,
12:36-39 (1992)) describe the detection and quantification of gene amplification products using a solid phase which is non-covalently coated with avidin. However, in that system hybridization is accomplished at 42° C. , a hybridization temperature often too moderate for high stringency hybridization.
Accordingly, what is needed in the art is a solid-phase hybridization apparatus which is tolerant of high-temperature and high stringency hybridization conditions and which can be readily, quickly, and inexpensively adapted to detect a variety of nucleic acid targets. The present invention provides these and other advantages.
SUMMARY OF THE INVENTION
In one aspect the present invention relates to a method of determining the presence or absence of a nucleic acid analyte from a sample. The method comprises the steps of binding a biotinylated capture oligonucleotide to the solid phase which is non-covalently coated with streptavidin or an avidin derivative. Simultaneous with, prior to, or subsequent to the binding step, the capture oligonucleotide is contacted with the nucleic acid analyte under stringent hybridization conditions with a temperature of at least 50° C. Under stringent hybridization conditions the capture oligonucleotide selectively hybridizes with the analyte to form a hybridization complex. The complex is detected as an indication of the presence or absence of said analyte in said sample.
In one embodiment, the solid phase is coated with avidin or streptavidin. In another embodiment, binding of the capture oligo and contacting the capture with the nucleic acid analyte are performed simultaneously. In a preferred embodiment, the solid phase is polystyrene. Preferably, the capture oligonucleotide is bound to the solid phase by a spacer of at least 36 covalent bonds in length. In another embodiment, stringent hybridization conditions are performed at between 55° C. and 70° C., or with a hybridization buffer having a chaotrope concentration of at least 1 molar. Preferably, the chaotrope is guanidine thiocyante. In another embodiment, the hybridization complex is detected by way of labelled primers used for nucleic acid analyte amplification.
In another aspect, the present invention is directed to a method of determining the presence or absence of a nucleic acid analyte from a sample. The method comprises the steps of binding a biotinylated capture oligonucleotide to the solid phase which is non-covalently coated with streptavidin or an avidin derivative. Simultaneous with, prior to, or subsequent to the binding step, the capture oligonucleotide is contacted with the nucleic acid analyte under stringent hybridization conditions with a temperature of at least 25° C. and a chaotrope concentration of at least 1 molar. Under stringent hybridization conditions the capture oligonucleotide selectively hybridizes with the analyte to form a hybridization complex. The complex is detected as an indication of the presence or absence of said analyte in said sample. In some embodiments the chaotrope is guanidine thiocyanate. In other embodiments the temperature is at least 40°. In additional embodiments the chaotrope concentration is at least 2M. The present invention has utility as an inexpensive, uncomplicated solid phase assay which can be rapidly adapted to detect a wide variety nucleic acid analytes.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is directed to compositions and methods for determining the presence or absence of a nucleic acid analyte from a sample. Generally, the method comprises binding a biotinylated capture oligonucleotide to a solid phase non-covalently coated with streptavidin or an avidin derivative. The capture oligonucleotide is contacted with, and specifically hybridizes under stringent conditions to, a nucleic acid analyte to form a hybridization complex. The presence or absence of the hybridization complex is an indication of the presence or absence, respectively, of the nucleic acid analyte in the sample. The steps for binding, and formation of the hybridization complex can be carried out sequentially in any order, or simultaneously per the desired assay format.
Quite surprisingly, we have determined that streptavidin and avidin derivatives unexpectedly remain non-covalently bound to each other and to a hydrophobic solid support under the harsh conditions of high temperature and/or high concentrations of chaotrope. Accordingly, the present invention provides solid phase supports which obviate the requirement for covalent attachment of oligonucleotides or streptavidin to the support. In turn, the costs and complications attendant with covalent attachment are eliminated.
Definitions
Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al. (1994)
Dictionary of Microbiology and Molecular Biology
, second edition, Jo
Campbell Eggerton A.
Saigene Corporation
Townsend and Townsend / and Crew LLP
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